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      Noninvasive imaging of immune responses

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          Significance

          Tumors are often surrounded and invaded by bone marrow-derived cells. Imaging the infiltration of such immune cells into tumors may therefore be an attractive means of detecting tumors or of tracking the response to anticancer therapy. We show that it is possible to detect these cells noninvasively by positron emission tomography (PET) via the surface markers displayed by them. The ability to monitor the immune response in the course of therapy will enable early determination of the efficacy of treatment and will inform decisions as to whether treatment should be stopped or continued. Noninvasive monitoring could therefore change how therapies are applied and assessed, to the benefit of many patients.

          Abstract

          At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (V H) of a camelid heavy-chain only antibody] with 18F or 64Cu. Radiolabeled VHHs rapidly cleared the circulation ( t 1/2 ≈ 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund’s adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked.

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          Most cited references26

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          Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity.

          Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The reactions tolerate a broad range of functionality and proceed in high yield in organic solvents, water, cell media, or cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k2 2000 M-1 s-1). This fast reactivity enables protein modification at low concentration.
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            Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine.

            The positron emitter zirconium-89 ((89)Zr) has very attractive properties for positron emission tomography (PET) imaging of intact monoclonal antibodies (mAbs) using immuno-PET. This protocol describes the step-by-step procedure for the facile radiolabeling of mAbs or other proteins with (89)Zr using p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). First, Df-Bz-NCS is coupled to the lysine-NH(2) groups of a mAb at pH 9.0 (pre-modification), followed by purification using gel filtration. Next, the pre-modified mAb is labeled at room temperature by the addition of [(89)Zr]Zr-oxalic acid solution followed by purification using gel filtration. The entire process of pre-modification, radiolabeling and purification steps will take about 2.5 h.
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              The use of 18F-FDG PET in the diagnosis of cardiac sarcoidosis: a systematic review and metaanalysis including the Ontario experience.

              Cardiac sarcoidosis is a potentially fatal complication of sarcoidosis. The 1993 guidelines of the Ministry of Health, Labour, and Welfare (MHLW) of Japan have been used as the diagnostic gold standard and for comparison with imaging modalities. (18)F-FDG PET is not currently included in the guidelines. However, studies have shown promising data using (18)F-FDG PET. We conducted a systematic review of studies that evaluated the accuracy of (18)F-FDG PET for the diagnosis of cardiac sarcoidosis compared with MHLW guidelines. Data from a prospective Ontario provincial registry are also reported and included in the metaanalysis. PubMed, Embase, and the Cochrane Central Register of Controlled Trials were searched for studies that satisfied predetermined criteria. Quality evaluation using the Quality Assessment for Diagnostic Accuracy Studies was performed by 2 independent masked observers. Data were extracted and analyzed to measure study-specific and pooled accuracy for (18)F-FDG PET compared with the MHLW as the reference. A total of 519 titles was identified; 7 studies, including the Ontario registry, were selected for inclusion. Metaanalysis of these 7 studies was conducted, with a total of 164 patients, most of whom had been diagnosed with systemic sarcoidosis. The prevalence of cardiac sarcoidosis was 50% in the whole population. Pooled estimates for (18)F-FDG PET yielded 89% sensitivity (95% confidence interval [CI], 79%-96%), 78% specificity (95% CI, 68%-86%), a 4.1 positive likelihood ratio (95% CI, 1.7-10), and a 0.19 negative likelihood ratio (95% CI, 0.1-0.4). The overall diagnostic odds ratio was 25.6 (95% CI, 7.3-89.5), and the area under the summary receiver operator characteristic curve was 93% ± 3.5. The Ontario study yielded sensitivity and specificity of 79% and 70%, respectively. The high diagnostic accuracy determined for (18)F-FDG PET in this metaanalysis suggests potential value for diagnosis of cardiac sarcoidosis compared with the MHLW guidelines. These results may affect patient care by providing supportive evidence for more effective use of (18)F-FDG PET in the diagnosis of cardiac sarcoidosis. Large-scale multicenter studies are required to further evaluate this role.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                12 May 2015
                20 April 2015
                20 April 2015
                : 112
                : 19
                : 6146-6151
                Affiliations
                [1] aWhitehead Institute for Biomedical Research , Cambridge, MA 02142;
                [2] bDepartment of Biology, Massachusetts Institute of Technology , Cambridge, MA 02142;
                [3] cCenter for Systems Biology, Massachusetts General Hospital , Boston, MA 02114;
                [4] dDepartment of Radiology, Massachusetts General Hospital , Boston, MA 02114;
                [5] eCenter for Immune Regulation, Oslo University Hospital, University of Oslo , N-0372 Oslo, Norway;
                [6]and fDepartment of Systems Biology, Harvard Medical School , Boston, MA 02115
                Author notes
                2To whom correspondence should be addressed. Email: ploegh@ 123456wi.mit.edu .

                Edited by Owen N. Witte, Howard Hughes Medical Institute, University of California, Los Angeles, CA, and approved March 17, 2015 (received for review February 18, 2015)

                Author contributions: M.R., E.J.K., A.M.B., and H.L.P. designed research; M.R. synthesized the substrates, expressed sortase and VHHs, and modified VHHs with fluorophores and for 64Cu/ 18F labeling; E.J.K. performed synthesis of 18F/ 64Cu-labeled VHH and their characterizations; M.R., E.J.K., G.R.W., and R.W. performed PET-CT imaging and analysis of the PET-CT data; M.R., A.M.B., J.T.J., and G.D.V. performed and interpreted two-photon microscopy; M.R., A.M.B., and J.N.D. performed FACS experiments; J.N.D., J.C., and L.K.S. identified VHH7 and DC13; and M.R. and H.L.P. wrote the paper.

                1M.R. and E.J.K. contributed equally to this work.

                Article
                201502609
                10.1073/pnas.1502609112
                4434737
                25902531
                75abdf8d-a821-49de-9732-14dfb6eb52a7

                Freely available online through the PNAS open access option.

                History
                Page count
                Pages: 6
                Funding
                Funded by: HHS | National Institutes of Health (NIH) 100000002
                Award ID: AI87879-06
                Categories
                Biological Sciences
                Immunology and Inflammation
                From the Cover

                pet imaging,non-invasive imaging,inflammation,cancer,camelid single domain antibodies

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